The metabolism of niacytin in the rat. Studies of the excretion of nicotinic acid metabolites

1. Nicotinic acid-deficient rats were given a dose of niacytin or an equivalent one of free nicotinic acid or hydrolysed niacytin. 2. The excretion of N'-methylnicotinamide and of tertiary nicotinoyl derivatives in urine showed that niacytin was not metabolized as free nicotinic acid, although hydrolysed niacytin was equivalent to free nicotinic acid. 3. Little or none of the niacytin dose was recovered as tertiary nicotinoyl derivatives in faeces. This result was not affected by fitting rats with tail-cups to prevent coprophagy. 4. At the high doses used niacytin restored the growth rate of the deficient animals because of a small degree of hydrolysis of the bound nicotinic acid.     

The metabolic origin of trigonelline in the rat.

A hypothesis of Mason & Kodicek [(1970) Biochem. J. 120, 515-521] that esterified nicotinic acid in niacytin from cereals is a precursor for trigonelline was investigated in rats. Single oral doses of niacytin resulted in the excretion of trigonelline in urine but only in rats that were niacin-deficient and were fed a cereal diet. These animals were found to have an abnormally permeable intestine, which allowed the uptake of molecules not usually absorbed. Orally administered synthetic [14C]nicotinoyl[3H]methylcellulose was shown to be absorbed by niacin-deficient rats on a cereal diet and [14C]trigonelline was excreted in urine. These data indicate that dietary cereal induces a permeability defect in the intestinal mucosa of niacin-deficient rats, which allows the uptake of macromolecular niacytin. The nicotinoyl pyridine nitrogen atom is then methylated and slow hydrolysis releases trigonelline from the macromolecule.     

Changes in trigonelline (N-methylnicotinic acid) content and nicotinic acid metabolism during germination of mungbean (Phaseolus aureus) seeds

Changes in trigonelline content and in biosynthetic activity were determined in the cotyledons and embryonic axes of etiolated mungbean (Phaseolus aureus) seedlings during germination. Accumulation of trigonelline (c. 240 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds; trigonelline content decreased 2 d after imbibition. Trigonelline content in the embryonic axes increased with seedling growth and reached a peak (c. 380 nmol per embryonic axis) at day 5. Trigonelline content did not change significantly during the differentiation of hypocotyls, and the concentration was greatest in the apical 5 mm. Nicotinic acid and nicotinamide were better precursors for pyridine nucleotide synthesis than quinolinic acid, but no great differences were found in the synthesis of trigonelline from these three precursors. Trigonelline synthesis was always higher in embryonic axes than in cotyledons. Activity of quinolinate phosphoribosyltransferase (EC 2.4.2.19), nicotinate phosphoribosyltransferase (EC 2.4.2.11), and nicotinamidase (EC 3.5.1.19) was found in cotyledons and embryonic axes, but no nicotinamide phosphoribosyltransferase (EC 2.4.2.12) activity was detected. It follows that quinolinic acid and nicotinic acid were directly converted to nicotinic acid mononucleotide by the respective phosphoribosyltransferases, but nicotinamide appeared to be converted to nicotinic acid mononucleotide after conversion to nicotinic acid. Trigonelline synthase (nicotinate N-methyltransferase, EC 2.1.1.7) activity increased in the embryonic axes, but decreased in cotyledons during germination. [14C]Nicotinic acid and trigonelline absorbed by the cotyledons were transported to the embryonic axes during germination. Trigonelline had no effect on the growth of seedlings, but nicotinic acid and nicotinamide significantly inhibited the growth of roots. Based on these findings, the role of trigonelline synthesis in mungbean seedlings is discussed.     

Trigonelline Content in Coffee Beans and the Thermal Conversion of Trigonelline into Nicotinic Acid during the Roasting of Coffee Beans

The trigonelline content in coffee was determined by the microbiological assay method after demethylating the compound. The content was proved to be extremely high (up to 1 % on the wet basis). When trigonelline was heated to above 180°C, it was converted into nicotinic acid. Although the conversion rate was low, a nutritionally significant amount of nicotinic acid was formed during roasting coffee beans because of the high content of trigonelline in coffee beans. The optimum heating condition for nicotinic acid formation was found at 220°C for 20 min.     

Metabolic fate of nicotinamide in higher plants

Metabolism of [carbonyl-¹⁴C]nicotinamide was surveyed in various plant materials including the model plants, Arabidopsis thaliana , Oryza sativa and Lotus japonicus . In all plants studied, nicotinamide was used for the pyridine (nicotinamide adenine) nucleotide synthesis, probably after conversion to nicotinic acid. Radioactivity from [carbonyl-¹⁴C]nicotinamide was incorporated into trigonelline (1- N -methylnicotinic acid) and/or into nicotinic acid 1 N -glucoside (Na-Glc). Trigonelline is formed mainly in leaves and cell cultures of O. sativa and L. japonicus and in seedlings of Trifolium incarnatum , Medicago sativa and Raphanus sativus . Trigonelline synthesis from nicotinamide is generally greater in leaves than in roots. Na-Glc was formed as the major nicotinic acid conjugate in A. thaliana and in tobacco Bright Yellow-2 cells. In seedlings of Chrysanthemum coronarium and Theobroma cacao , both trigonelline and Na-Glc were synthesized from [carbonyl-¹⁴C]nicotinamide. Trigonelline is accumulated in some seeds, mainly Leguminosae species. The pattern of formation of the nicotinic acid conjugates differs between species and organs.     

The metabolism of labelled hexadecyl sulphate salts in the rat, dog and human.

The metabolic fate of [1-14-C]hexadecylsulphate and hexadecyl[35-S]sulphate, administered intravenously as the sodium and trimethylammonium salt to dogs and orally as the erythromycin salt to dogs, rats and humans, was studied. Studies with rats indicated that the compounds were well absorbed and rapidly excreted in the urine. However, after oral administration of the 14-C-and 35-S-labelled hexadecyl sulphate erythromycin salt to dogs, considerable amounts of radioactivity were excreted in the faeces as unmetabolized hexadecyl sulphate. Studies with two humans showed that orally administered erythromycin salt of [1-14C]hexadecyl sulphate was well absorbed in one person but poorly absorbed in the other. Radioactive metabolites in urine were separated by t.l.c. in two solvent systems. The main metabolite of hexadecyl sulphate in the dog, rat and human was identified as the sulphate ester of 4-hydroxybutyric acid. In addition, psi-[14-C]butyrolactone as a minor metabolic product of [1-14-C]hexadecyl sulphate was also isolated from the urine of rat, dog and man. However, there was still another metabolite in dog urine, which comprised about 20% of the total urinary radioactivity and carried both 14-C and 35-S labels. This metabolite was absent from rat urine. The metabolite in dog urine was isolated and subsequently identified by t.l.c. and g.l.c. and by isotope-dilution experiments as the sulphate ester of glycollic acid. Small amounts (about 5% of the total recovered radioactivity in excreta) of labelled glycollic acid sulphate were also found in human urine after ingestion of erythromycin [1-14-C]hexadecyl sulphate.     

Pyridine nucleotide cycle and trigonelline (N-methylnicotinic acid) synthesis in developing leaves and fruits of Coffea arabica

We examined the biosynthesis of trigonelline in leaves and fruits of Arabica coffee ( Coffea arabica ) plants. [³H]Quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [¹⁴C]nicotinamide and [¹⁴C]nicotinic acid, which are degradation products of NAD, were converted to trigonelline and pyridine nucleotides. These tracer experiments suggest that the pyridine nucleotide cycle, nicotinamide → nicotinic acid → nicotinic acid mononucleotide (NaMN) → nicotinic acid adenine dinucleotide (NaAD) → NAD → nicotinamide mononucleotide (NMN) → nicotinamide, operates in coffee plants, and trigonelline is synthesized from nicotinic acid formed in the cycle. Trigonelline accumulated up to 18 µmol per leaf in developed young leaves, and then decreased with age. Although the biosynthetic activity of trigonelline from exogenously supplied [¹⁴C]nicotinamide was observed in aged leaves, the endogenous supply of nicotinamide may be limited, reducing the contents in these leaves. Trigonelline is synthesized and accumulated in fruits during development. The trigonelline synthesis in pericarps is much higher than that in seeds, but its content in seeds is higher than pericaps, so that some of the trigonelline synthesized in the pericarps may be transported to seeds. Trigonelline in seeds may be utilized during germination, as its content decreases. Trigonelline synthesis from [¹⁴C]nicotinamide was also found in Theobroma cacao plants, but instead of trigonelline, nicotinic acid-glucoside was synthesized from [¹⁴C]nicotinamide in Camellia sinensis plants.     

Metabolism of nicotinic acid in plant cell suspension cultures, III: Formation and metabolism of trigonelline (author's transl)]

Cell suspension cultures of Phaseolus aureus, Glycinemax., Cicer arietinum and Chenopodium rubrum convert nicotinic acid and nicotinamide into N-methyl nicotinic acid (trigonelline). Application of [carboxyl-14C]- and [N-methyl-14C]nicotinic acid to cell cultures demonstrated that 1) the nicotinic acid moiety of trigonelline is funnelled into the pyridine nucleotide cycle, 2) trigonelline is demethylated partly oxidatively, but predominantly non-oxidatively, transferring the methyl carbon atom to still unknown acceptors, and 3) uptake of trigonelline by mung bean cell cultures is accompanied by demethylation and instantaneous remethylation reactions. Cell suspension cultures of parsley (Petroselinum hortense Hoffm.) show uptake but no metabolism of trigonelline. The data are compared with trigonelline metabolism in intact plants.     

The metabolism of N-triphenylmethylmorpholine in the dog and rat

1. Rats were given N-triphenyl[(14)C]methylmorpholine, triphenyl[(14)C]carbinol, N-triphenylmethyl[G-(3)H]morpholine or [G-(3)H]morpholine as single oral doses; the routes of excretion were examined. 2. Dogs were given single oral doses of N-triphenyl[(14)C]methylmorpholine. 3. (14)C-labelled metabolites were excreted mainly in the faeces in both rats and dogs; no (14)CO(2) was expired and less than 3% remained in the carcass and skin after 96hr. 4. (3)H-labelled metabolites were excreted rapidly in urine; part of the label was found in the expired gases and over 10% remained in the carcass and skin after 96hr. 5. Differences in excretion pattern between the sexes were noticed in rats but not in dogs. 6. N-Triphenylmethylmorpholine was rapidly hydrolysed to form triphenylcarbinol and morpholine in the stomach; morpholine was absorbed rapidly and excreted largely unchanged, though some was degraded, since some of the (3)H was found in water. 7. Triphenylcarbinol was absorbed only slowly and was oxidized to p-hydroxyphenyldiphenylcarbinol. 8. Both triphenylcarbinol and its p-hydroxy derivative were found in urine, bile and faeces in the free form and conjugated with glucuronic acid. The proportion of conjugates was higher in rat bile than in faeces. 9. Traces of o-hydroxyphenyldiphenylcarbinol and m-hydroxyphenyldiphenylcarbinol were detected as metabolites both free and conjugated.     

Nicotinic acid and nicotinamide metabolism and promotion of cell arrest in G2 in Pisum sativum

Nicotinic acid and nicotinamide are immediate precursors of trigonelline, a hormone present in cotyledons of Pisum sativum L. which promotes cell arrest in G2 during cell maturation in roots and shoots. All three compounds are members of the pyridine nucleotide pathway for the synthesis of NAD and NADP. Concentrations of nicotinic acid and nicotinamide in excised roots grown for 3 days in White's medium with sucrose were determined by HPLC. Results suggest that nicotinamide is rapidly converted first to nicotinic acid and then trigonelline. High nicotinic acid concentrations may occur in excised roots. Conversion of trigonelline to nicotinic acid in excised roots did not occur in these experiments. The concentrations of either nicotinamide or nicotinic acid in roots are not related to the proportions of cells arrested in G2. Trigonelline promotes cell arrest in G2, and nicotinic acid and nicotinamide are active only because they are converted to trigonelline.     

Alteration of urinary levels of the carcinogen, N-hydroxy-2-naphthylamine, and its N-glucuronide in the rat by control of urinary pH, inhibition of metabolic sulfation, and changes in biliary excretion

The hepatic metabolism of arylamine bladder carcinogens to N-hydroxy arylamine N-glucuronides, their excretion in the urine, and their subsequent acidic hydrolysis to highly carcinogenic and reactive N-hydroxy arylamines have been proposed as essential steps in arylamine-induced urinary bladder carcinogenesis. In this study, alteration of urinary pH, inhibition of metabolic sulfation, and blockage of biliary disposition were shown to profoundly affect the urinary excretion of the probable ultimate bladder carcinogen, N-hydroxy-2-naphthylamine (N-HO-2-NA) and its N-glucuronide conjugate. The normal pH of rat urine (6.7) was altered to 5.7 or 7.7 by administration of NH4Cl or NaHCO3 in the drinking water. Subsequent treatment with either 2-naphthylamine (2-NA) or 2-nitronaphthalene (2-NN) resulted in increased urinary levels of free N-HO-2-NA (relative to its N-glucuronide) in acidic urines and decreased relative amounts of free N-HO-2-NA in alkaline urines. In addition, 2-NN yielded 5--10-fold greater levels of urinary N-HO-2-NA and its N-glucuronide than rats given 2-NA; and 2-NA was not detected as a urinary metabolite of 2-NN. Some 12 additional metabolites of 2-NA and 2-NN were also found. Of these, 2-amino-1-naphthol and its sulfate and glucuronide conjugates were quantitated. From these data, 2-NA and 2-NN appear to share common metabolic pathways which yield free N-HO-2-NA as a putative ultimate urinary bladder carcinogen. Pentachlorophenol, a known inhibitor of hepatic sulfotransferases, was shown to cause a 2--3-fold increase in the urinary levels of N-HO-2-NA N-glucuronide and N-HO-2-NA from 2-NA-treated rats. Similarly, inhibition of the biliary excretion of 2-NA by bile duct ligation resulted in a 6-fold increase in total urinary N-HO-2-NfA. Furthermore, analyses of bile revealed that substantial amounts of N-HO-2-NA N-glucuronide, but not free N-HO-2-NA, were present. The role of urinary versus biliary excretion of N-hydroxy arylamines in relation to bladder and colon carcinogenesis is discussed.     

Urinary kallikrein excretion after potassium adaptation in the rat

24-h urinary kallikrein excretion in male Sprague-Dawley rats was measured before and after 14 days with 100 mM potassium chloride as drinking fluid ad libitum. Urinary kallikrein excretion increased in K+-adaptation. The increase was greater when the rats were given distilled water rather than 100 mM sodium chloride to drink prior to the potassium chloride. The urinary potassium excretion increased in all rats studied. The urinary sodium excretion, urine volume and fluid intake increased significantly in rats that had distilled water to drink prior to the KCl. In marked contrast, when rats were offered NaCl prior to KCl, the urinary sodium excretion was unaffected while the urine volume and fluid intake decreased significantly. This study shows that prior NaCl intake abolishes the natriuretic and diuretic effects of KCl load and only suppresses the increase in urinary kallikrein excretion. This suggests that K+ secretory activity at the distal tubules is the major determinant of the release of renal kallikrein in the rat.     

Excretion of catecholamines in rats,mice and chicken

Stress assessment favours methods, which do not interfere with an animal's endocrine status. To develop such non-invasive methods, detailed knowledge about the excretion of hormone metabolites in the faeces and urine is necessary. Our study was therefore designed to generate basic information about catecholamine excretion in rats, mice and chickens. After administration of (3)H-epinephrine or (3)H-norepinephrine to male and female rats, mice and chickens, all voided excreta were collected for 4 weeks, 3 weeks or for 10 days, respectively. Peak concentrations of radioactivity appeared in one of the first urinary samples of mice and rats and in the first droppings in chickens 0.2-7.2 h after injection. In rats, between 77.3 and 95.6% of the recovered catecholamine metabolites were found in the urine, while in mice, a mean of 76.3% were excreted in the urine. Peak concentrations in the faeces were found 7.4 h post injection in mice, and after about 16.4 h in rats (means). Our study provides valuable data about the route and the profile of catecholamine excretion in three frequently used species of laboratory animals. This represents the first step in the development of a reliable, non-invasive quantification of epinephrine and norepinephrine to monitor sympatho-adrenomedullary activity, although promising results for the development of a non-invasive method were found only for the chicken.     

The urinary excretion of orally administered pteroyl-l-glutamic acid by the rat

1. The urinary excretion of folates after oral administration of [2-(14)C]pteroyl-l-glutamic acid was studied by assaying the radioactivity in the urine and in materials purified and characterized by t.l.c. 2. Radioactivity excreted was 6.8, 5.9 and 30.7% of the oral dose in the first 24h after doses of 3.1, 32 and 320mug/kg respectively. 3. Extensive decomposition of urinary folates to pteroyl-l-glutamic acid was prevented by antioxidants or collection of urine frozen. 4. At the three dosages, two major and one minor radioactive compounds were isolated. One of the major metabolites was 5-methyltetrahydropteroylglutamic acid. The others were unidentified but were not pteroylglutamic acid, 7,8-dihydro-, 5,6,7,8-tetrahydro-, 5- or 10-formyl-tetrahydro-, 5,10-methylidyne-tetrahydro-, 5-formimidoyl-tetrahydro-, 5,10-methylene-tetrahydro-, 5-methyltetrahydro-pteroylglutamic acid, nor any decomposition products of these compounds formed during isolation. Labelled unconjugated pteridines were absent. 5. Labelled pteroyl-l-glutamic acid was displaced by oral administration of unlabelled pteroyl-l-glutamic acid (1.6mg/kg) when given 3.5h after, but not when given 24h after the labelled dose. 6. The results show that orally administered [2-(14)C]pteroyl-l-glutamic acid is absorbed without metabolism and is then metabolized into naturally occurring tetrahydro-folates. 7. These findings are discussed with reference to previous work.     

垂体在刺激脑内渗透压感受器引起的肾脏反应中的作用

实验在α氯醛糖和氨基甲酸乙酯混合麻醉的大鼠中进行。脑室内注射高张盐水(icv.HS)后,肾血浆流量、肾小球滤过率、尿量、尿钠排出量、尿钾排出量和渗透物质清除率均增加,游离水清除率下降。去除垂体后,icv.HS不再能引起上述肾脏反应。另外给大鼠静脉注射血管升压素(VP)拮抗剂(V_1和V_2受体拮抗剂),并不能削弱上述icv.HS引起的肾脏反应。脑室内注射高张盐水后,尿中多巴胺(DA)排出量无显著增多;给予多巴脱羧酶抑制剂苄丝肼也不能削弱icv.HS引起的肾脏反应。上述实验结果表明,在本实验条件下刺激脑内渗透压感受器引起的肾脏反应依赖于垂体的完整性,但看来并不依赖于外周的VP和DA,故垂体通过何种机制介导icv.HS引起上述肾脏反应,有待于进一步的研究。     

Mechanism of enhanced bioavailability and diuretic effect of azosemide by ascorbic acid in rats

To increase the extent of comparative oral bioavailability (F) value and the diuretic and natriuretic effects of orally administered azosemide, ascorbic acid was coadministered to rats. The rationales for this study are that ascorbic acid might inhibit intestinal first-pass effect of azosemide and might increase the unionized fraction of azosemide at the receptor sites. After oral administration of azosemide (20 mg/kg) with 100 mg of ascorbic acid, the F value (138% vs. 100%), 8-h urinary excretion of azosemide (5.18% vs. 1.32% of oral dose), 8-h urine output (41.3 vs. 23.0 ml), and 8-h urinary excretion of sodium (24.6 vs. 15.3 mmol/kg) were greater than controls (without ascorbic acid). The amount of spiked azosemide remaining after 30 min incubation of 50 mug of azosemide with the 9000 g supernatant fraction of rat small intestine was significantly greater by 100 microg of ascorbic acid (45.3 vs. 40.9 microg) than controls (without ascorbic acid). After oral administration of azosemide with NH4Cl, the urine pH decreased by 0.5 U, and 8-h urine output (25.8 vs. 11.0 ml) and 8-h urinary excretion of sodium (13.3 vs. 6.89 mmol/kg) were significantly greater than controls (without NH4Cl). The increase in F value and diuretic and natriuretic effects of azosemide with coadministration of ascorbic acid seemed to be due to reduced intestinal first-pass metabolism of azosemide, increased urinary excretion of azosemide, and increased unionized fraction of azosemide at the renal tubular receptor sites.     

Metabolism of the herbicide 2,6-dichlorobenzonitrile in rabbits and rats

1. The metabolism of 2,6-dichlorobenzonitrile was studied in rabbits and rats. Oral administration caused an increased urinary excretion of glucuronides and ethereal sulphates. There was also an indication of mercapturic acid formation. 2,6-Dichloro-3-hydroxybenzonitrile and its 4-hydroxy analogue were identified as metabolites in the urine. A small amount of the unchanged substance was recovered from the faeces. 2. By using 2,6-dichlorobenzo[¹⁴C]nitrile the phenolic metabolites were determined quantitatively and some other possible metabolic routes were excluded. 3. Incubation of 2,6-dichlorobenzonitrile with enzyme preparations (papain and high-speed supernatant of rat-liver homogenate plus glutathione) gave no indications for a reaction with thiol compounds.     

Metabolic disposition of [14C] metanil yellow in rats

The absorption, metabolism and excretion of [14C] metanil yellow was studied in rats. Following administration of a single oral dose of 5 mg dye (7.6 microCi)/kg body weight, 80.5% of the dose was excreted in the urine and faeces within 96 hr, with the majority being accounted for in the faeces. Liver, kidney, spleen and testis retained no count whereas 13.6% of the radioactivity was retained by gastrointestinal tract. Analysis of urine and faeces detected two azo-reduction metabolites of metanil yellow which were characterized by TLC and IR, NMR and mass spectroscopic studies as metanilic acid and p-aminodiphenylamine.     

The diuretic effect of metipamide and its relationship to body weights in rats

Metipamide [M], a new Czechosclovak diuretic with a hypotensive effect, was administered in a dose of 20 mg/kg (about 500-fold the therapeutic dose) I. for three weeks to rats of both sexes kept under normal conditions in groups of five, and II, for eight days to single male rats in metabolic cages. The animals' body weight and food and water consumption were studied and in the second series their daily faeces, urine and urinary sodium and potassium excretion were measured. Rats kept in individual cages were also given indapamide (I), the first diuretic with a separate hypotensive effect used in other countries, in a dose of 20 mg/kg. The experimental animals' body weight was significantly lower than that of controls with the same food consumption and their water intake and urine flow were much higher, especially after M. Sodium (and to a lesser extent potassium) excretion was raised at the outset of administration of both the test substances and again after the 5th to 8th dose, but only after M. After three weeks' administration of M. SNa, SK and S(osm) values were within normal limits. but after eight days the serum electrolytes and the osmolality of the serum were markedly reduced. After I. these values were normal. We conclude that the strong diuretic effect of M is not the only cause of lower body weight in rats.     

Influence of renal denervation on chronopharmacology of furosemide in rats.

Our previous studies have suggested that the adrenergic nervous system is involved in the mechanism responsible for the time-dependent change in the urinary excretion of furosemide in rats. To examine a potential role of renal nerves in this phenomenon, renal denervation or sham operation was performed using unilaterally nephrectomized rats. Furosemide (30 mg/kg) was given orally at 12 am or 12 pm. Urine was collected for 8 hours after furosemide dosing, and urinary excretions of furosemide and sodium were determined. Urinary furosemide excretion and diuretic effects of the agent (urine volume and urinary sodium) were significantly greater at 12 am than at 12 pm in the sham-operated group of rats. However these administration time-dependent changes in urinary furosemide and its diuretic effects disappeared in the renal-denervated group of animals. These results suggest that the renal nerves contribute to the time-dependent changes in the urinary excretion of furosemide and its subsequent diuretic effects.